The broad purpose of the research in this proposal is to determine the fundamental relationships between the structures of active sites in metalloproteins and function. A bio-inspired synthetic approach is utilized that incorporates structural components found in active sites, specifically those that control the microenvironment (secondary coordination sphere) surrounding metal ion(s). Organic compounds are being developed which create rigid structures around a bound metal ion, forming specific microenvironments that emulate motifs found within protein active sites. Regulation is achieved via non- covalent interactions (e.g., hydrogen bonds) that are strategically placed proximal to the metal ions to affect function; in particular the binding and activation of dioxygen, which are processes directly linked to the maintenance of human health and aging. These synthetic systems allow for the stabilization of high-energy transients that are often proposed, but rarely observed, in non-heme metalloenzymes: their detection provides new avenues to explore mechanistic aspects of these biological essential reactions. Long-term goals include developing structure function relationships in metal-assisted oxidative catalysis. Metalloproteins perform functions not yet achieved in synthetic systems. Our hypothesis is that the lack of control of the secondary coordination sphere in synthetic compounds is a major obstacle to desired functions. Results from structural biology show that non-covalent interactions within the secondary coordination spheres of metalloproteins are instrumental in regulating function. Therefore the function and dysfunction of health-related metalloproteins can be understood in the context of changes in their microenvironments. It is still unclear, even in biomolecules, how non-covalent interactions are able to influence metal-mediated processes. Investigations into these effects require basic reactivity and mechanistic studies in which the effects of single components can be analyzed individually. We have developed synthetic systems whereby site-specific modulations in structure can be readily accomplished, in order to establish correlations with function, these types of studies will lead to fundamental mechanistic insights into biochemical processes. Ultimately, this research will provide insights into the properties of biological catalysts and lead to new classes of synthetic catalysts that incorporate the exquisite control of reactivity characteristic of metalloenzymes.